Experiments proposed in this application have a two-fold purpose: first to develop detailed kinetic models for electrogenic secondary transport systems in biological membranes; and second, to describe and identify the principal cellular reactions by which metabolism controls both primary and secondary transport. (Control, in this sense, means to modulate according to immediate, integrated metabolic requirements; and must be distinguished both from supplying energy--as in the form of ATP, and from regulating--over the long term--via genetic mechanisms and protein synthesis). The model system to be used is that of the plasma membrane of the fungus Neurospora crassa, which--upon nutrient limitation--rapidly develops (derepresses) a variety of ion-dependent and membrane potential-dependent uptake systems: for carbohydrates, amino acids, inorganic anions, and inorganic cations, particularly ammonium; which also--under special conditions of respiratory limitation--displays oscillations of membrane potential of a form requiring both primary (ATP-dependent) and secondary transport systems to be controlled; and which admits direct determination of membrane electrical properties with microelectrodes. The first objective will be approached by making detailed flux-kinetic measurements on the derepressible high-affinity transport systems for ammonium ions and glucose, and combining these results with complementary electrical results obtained from current-voltage curve measurements. The second objective will be approached from three directions: detailed flux measurements, to identify those discrete transport systems which do oscillate; relaxation experiments to identify and label (with time-constants) the major reactions connecting metabolism to the membrane potential; and extensive measurements of cellular levels and efflux of the substance believed to be the control "messenger": adenosine 3',5'-cyclic monophosphate (cyclic AMP). Throughout the experiments use will be made of specialized mutants of the organism, including strains which are defective for particular transport systems, strains with an accessory respiratory pathway, and strains severely defective in cyclic AMP metabolism.